The Reactive Oxygen Species (ROS) such as the superoxide anion (O2−, the hydrogen peroxide (H2O2) and hydroxyl radical (OH), are generated as products of the normal aerobic cell metabolism. In physiological conditions there is a balance between the generation speed and the dissipation speed of the ROS. When the dissipation speed decreases or when the generation speed increases, the cell enters into a pro-oxidative or oxidative stress state. The increase of the ROS is associated with several pathologies, such as the arteriosclerosis, cardiovascular diseases, rheumatoid arthritis, neurodegenerative disorders and cancer (Valko M et al. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 39(1):44-84, 2007).
Several experimental evidence relate ROS with the cancer etiology for their mutagenic capacity as well as for their participation in signal transduction pathways related with the induction of the proliferation, angiogenesis, migration and metastasis (Wu W. The signaling mechanism of ROS in tumor progression. Can Met Rev 25:695-705, 2006). Bibliographic evidence shows that a permanent oxidative stress state may be related with the appearance of the malignant phenotype (Halliwell B. Oxidative stress and cancer: have we moved forward? Biochem, 402(1):1-11, 2007). It has been proved that there is a high production of H2O2 in different types of cancer, including melanoma, neuroblastoma, and breast, colon and pancreas cancer (Szatrowski, T., Nathan, C. Production of large amounts of hydrogen peroxide by human tumor cells. Cancer Res. 51: 794-798, 1991).
It has been proved that there is an association between the increase of the cell malignity and the increase in the production of free radicals (Policastro L., Molinari B., Larcher F., Blanco P., Podhajcer O. L., Costa C. S., Rojas P., Durán H. Imbalance of antioxidant enzymes in tumor cells and inhibition of proliferation and malignant features by scavenging hydrogen peroxide. Mol. Carcinog. 39(2): 103-13, 2004). In addition to this experimental evidence there are studies that show high levels of ROS in several types of cancerous tissue as compared with their normal counterparts (Toyokuni S. et al. Persistent oxidative stress in cancer. FEBS Lett. 358: 1-3, 1995).
Gene therapy represents a promissory therapeutic strategy, consisting on the introduction of genetic sequences in receptor cells in order to replace the defective genetic material or to confer a new cellular activity. Presently, this is one of the most important developments that are taking place in medicine. In order to modify a specific type of cell or tissue, therapeutic genes must be efficiently administered to the cell, so that the gene is expressed in the appropriate level and during a sufficient period of time. One of the most relevant requirements for the potential use of therapeutic genes in cancer—as well as in other diseases, is the selectivity of the expression of the therapeutic gene
The use of gene promoters that are differentially expressed in the tumor tissue respect of the normal tissue is one of the ways of attributing that specificity. (Sadeghi H and Hitt M. Transcriptionally targeted adenovirus vectors. Curr Gene Ther 5(4):411-427, 2005).
However, the expression of highly expressed genes in the cancerous tissue is frequently heterogeneous among the cells of the same tumor and even more between the different tumors. A way of approaching this problem is to use promoter sequences responsive to a differential feature of the malignant environment. In this respect, some properties of the tumor micro-environment, such as hypoxia, glucose consumption, acid environments and increased angiogenesis, have been explored as distinctive features of tumors. The presence of DNA motifs responsive to these features have been studied as possible specific cancer promoters for driving the expression of therapeutic genes (Xu G. et al. Strategies for enzyme/prodrug cancer therapy. Clin. Cancer Res. 7: 3314-3324, 2001). For example, several works have proved that hypoxia can be used for the differential gene expression, by using elements responsive to low oxygen tension called hypoxia responsive elements (HRE, Hypoxia Responsive Elements) (Shibata T, et al. Development of a hypoxia-responsive vector for tumor specific gene therapy. Gene Ther. 7: 493-498, 2000). However, more recent evidence indicates that hypoxic regions are not distributed homogenously in the tumor, therefore limiting the use of HRE (Ballinger J. Imaging hypoxia in tumor. Semin Nucl Med. 31(4):321-9, 2001).
Recent studies describe the presence of promoter sequences sensitive to the reactive oxygen species in promoters of several genes. Among the genes that are highly expressed in cancer and that have motifs sensitive to ROS in their promoters we may mention the vascular endothelial growth factor (VEGF), the Early Growth Response-1 gene (EGR-1) and the matrix metalloproteinase promoter-1 (MMP-1). The VEGF promoter has a region GC-rich in residues that can be regulated by variations in the cellular redox state (Schafer G. et al. Oxidative stress regulates vascular endothelial growth factorA gene transcription through Sp1 and Sp3 dependent activation of two proximal GC-rich promoter elements. JBC. 278: 8190-98, 2003). Besides, the EGR-1 promoter has motifs rich in A and T bases that form 10 bp motifs of CC (A/T)6GG bases (SEQ ID NO:29) or CArG domains (Datta R. et al. Reactive oxygen intermediates target CC(A/T)6GG sequences to mediate activation of the early growth response 1 transcription factor gene by ionizing radiation. Proc. Natl. Acad. Sci. 90: 2419-22, 1993). Finally, the MMP-1 promoter has a region comprised between bases −2002 and −1546 showing the response to the presence of ROS (Nelson K et al. Elevated Sod2 activity augments matrix metalloproteinase expression: evidence for involvement of endogenous hydrogen peroxide in regulating metastasis. Clin. Cancer Res. 9: 424-432, 2003).
US patent document 2003/0082685 to Weichselbaum et al discloses therapeutic methods for treating several types of cancer and hyperproliferative diseases, comprising the expression of a therapeutic gene driven by the EGR-1 promoter. It indicates that the therapeutic method comprises exposing the cell to ionizing radiation, inducing the EGR-1 promoter that therefore drives the expression of the genes under its control.
U.S. Pat. No. 5,830,880 to Sedlacek et al discloses a DNA construct for the prophylaxis or therapy of tumor diseases comprising an activator sequence, a cycle-regulated cell promoter module, and a DNA sequence codifying for an antitumor substance, such as an angiogenesis inhibitor, a cytostatic agent or an enzyme. A complete VEGF promoter is used, evaluating the response to hypoxia environments (low oxygen tension) of HRE motifs comprised in the VEGF promoter. The HRE motifs do not co-localize with the ROS-RE motifs.
The use of specific promoters is essential but not sufficient for a gene therapy to be effective, since it also requires for the definition of an effective therapeutic gene. One of the attractive modalities of gene therapy in cancer is the use of the suicide genes. The system basis consists of introducing an enzyme codifying gene capable of metabolizing a non-toxic pro-drug, converting the same in toxic. One of the genes mostly used is the herpes virus thymidine kinase or HSV-TK (Herpes Simples Virus thymidine kinase) that codifies for an enzyme capable of phosphorilating to the pro-drug acyclovir/ganciclovir (widely used antiviral in viral infections), an analogue to guanosine. The phosphorylated antiherpetic is incorporated to the DNA molecule, avoiding its duplication and causing cellular death (Mooften F. L., Drug sensitivity (“suicide”)genes for selective cancer chemotherapy, Cancer Gene Ther, 1:279-287, 1994). Neighboring tumor cells that have not incorporated the gene can also be eliminated by the called bystander effect, which allows for the toxic metabolites to transfer from an affected to an unaffected cell.
Koshikawa et al (Cancer Res. 60: 2936-2941, 2000) disclose the use of the complete VEGF promoter, comprising the HRE site, which when exposed to hypoxia conditions drives the HSV-TK expression in lung carcinoma cells, where its use increases the tumor regression. The HRE motifs do not co-localize with the ROS-RE motifs.
US patent application 2001/0006954 to Weichselbaum et al discloses a DNA molecule comprising an alfa tumor necrosis factor gene (and other therapeutic genes as cytokines, toxins, tumor suppressors, etc.,) under the transcriptional control of the CArG domains of the EGR-1 promoter that are inducible by ionizing radiation.